Complex glycans associated with the cell surface of bacteria play central roles in bacterial survival in the environment and in hosts. Those glycans provide resistance to environmental and host challenges, including detergents, host immune processes, and changes in osmolarity and pH. Historically it has been difficult to ascribe specific virulence functions to individual polysaccharides, and several conflicting reports describing these relationships exist. The primary reason for this lack of knowledge is the inherent difficulty in selectively detecting and quantifying specific bacterial polysaccharides, and the discovery that often disruptions in glycan biosynthesis pathways have complex effects related to the sequestration of a key common substrate, bactoprenyl phosphate. These problems manifest in both the biology of bacteria, and in synthetic biology schemes to heterologously express new glycans in new species. The major goal of this proposal is to develop tools to detect and quantify bactoprenyl phosphate to help understand how to optimize glycan expression based on substrate abundance, and to develop complementary tools to detect glycans on the surface of bacteria and in cell lysates. In this program we will focus on three key glycans: Campylobacter jejuni N-linked oligosaccharide, and Bacteroides fragilis capsular polysaccharide A. In specific aim one we focus our efforts on developing a system for the immobilization of chemoenzymatically and cell prepared glycans that can be used to investigate glycan binding partners from antibodies to lectins. Preliminary work with the C. jejuni N-linked oligosaccharide provides key background on our success in this area. In the second specific aim we investigate how the abundance of bactoprenyl phosphate impacts production of a recombinant glycan using new tools in LC-MS and the tools developed in aim 1. We also take an unbiased directed evolution approach to improving recombinant glycan production in E. coli. Using this information we will build an E. coli strain better optimized for recombinant glycan expression. Together this application provides a series of tools that can be used by microbiologists and analytical chemists for the investigation of critical systems in glycoscience that are important targets for new therapeutics. Modified Specific Aims In vitro and Cellular Tools for Complex Polysaccharide Biosynthesis Bacterial surface polysaccharides play central roles in a wide range of biology and could serve as targets for novel anti-microbial agents, pathogen sensors, vaccine antigens, or other important therapeutics. Some bacterial surface polysaccharides, including the Bacteroides fragilis polymer capsular polysaccharide A (CPSA), a major focus of this proposal, could also serve as therapeutics themselves. These applications require robust methods to produce these materials that can be easily adapted from one type of polysaccharide to another. A majority of bacterial polysaccharides are prod...